Bioresource Technology 167 (2014) 358–366
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Cell growth kinetics of Chlorella sorokiniana and nutritional values of its biomass Kanhaiya Kumar, Chitralekha Nag Dasgupta, Debabrata Das ⇑ Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India
h i g h l i g h t s Physico-chemical parameters were optimized for the growth of C. sorokiniana. Arrhenius growth activation energy was calculated to show its fast growing nature. Monod model was used to study the growth kinetics of this microorganism. Stoichiometric analysis was performed to measure CO2 intake and biomass synthesis. Astaxanthin and b-carotene were separated from chlorophyll contamination.
a r t i c l e
i n f o
Article history: Received 11 May 2014 Received in revised form 29 May 2014 Accepted 31 May 2014 Available online 16 June 2014 Keywords: Temperature pH Light Elemental analysis Pigment extraction
a b s t r a c t The present study investigates the effects of different physico-chemical parameters for the growth of Chlorella sorokiniana and subsequently determination of nutritional values of its biomass. Most suitable temperature, light intensity, pH, and acetic acid concentration were 30 °C, 100 lmol m2 s1, pH 7.5, and 34.8 mM, respectively for the growth of this microorganism. Arrhenius growth activation energy, Ea was calculated as 7.08 kJ mol1. Monod kinetics constants: maximum specific growth rate (lmax) and substrate (acetic acid) affinity coefficient (Ks) were determined as 0.1 ± 0.01 h1 and 76 ± 8 mg L1, respectively. Stoichiometric analysis revealed the capture of 1.83 g CO2 and release of 1.9 g O2 for 1 g algal biomass synthesis. Algal biomass of C. sorokiniana was found rich in protein and several important minerals such as Mg, Ca, and Fe. Astaxanthin and b-carotene were extracted and quantified using high performance liquid chromatography. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction World is facing several challenges such as inadequate availability of food, feed and fuels. Global warming due to greenhouse gases like, CO2 is a major concern (Kumar and Das, 2014). Exploring the algal technology is a promising area, which can support the supply of these demands. Microalgae can transform light energy into chemical energy to meet the future energy demand (e.g. biodiesel, biohydrogen) and they can produce a great variety of useful metabolites (Kumar et al., 2013; Xin et al., 2011). It is a promising source of several pigments such as lutein, b-carotene, and astaxanthin (Kumar et al., 2014; Cordero et al., 2011). These pigments are known for their excellent antioxidant and pharmaceutical properties. In addition, algal biomass has high nutritional values as it is
⇑ Corresponding author. Tel.: +91 03222 283758; fax: +91 3222 255303. E-mail address: [email protected] (D. Das). http://dx.doi.org/10.1016/j.biortech.2014.05.118 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
rich source of protein, carbohydrate, essential fatty acids, dietary fibers, minerals etc. Different physio-chemical parameters such as pH, temperature, light, different nutrients are governing the activities and growth rates of microalgae when they are grown in different carbon sources like CO2 and acetic acid. At low light intensity, photosynthesis becomes the limiting factor. The light intensity above the saturation level inhibits the growth by photoinhibition and excess excitation energy is dissipated as fluorescence or heat through non-photochemical quenching (NPQ) (Yamakawa and Itoh, 2013). The low temperature reduces many cellular activities such as the ability to use light energy by limiting the electron transport chain etc. (Falk et al., 1990). Photosystem II is also very thermolabile, causing inhibition of photosynthesis at higher temperature (Fork et al., 1979). Photorespiration was found to increase with the increase in temperature (Berry and Raison, 1981). In addition, temperature can also affect the cytosolic pH and other physical processes (Raven and Geider, 1978). Mostly the microalgae
K. Kumar et al. / Bioresource Technology 167 (2014) 358–366
maintain neutral or slightly alkaline cytosolic pH, as many enzymes are highly pH dependent and become inactive in acidic pH (Gimmler, 2001). Therefore, external pH plays important role for maintaining the cytosolic pH. In addition, it has been found that pH is the major determinant for the availability of the carbonaceous species in the broth for the growth of algae. High alkaline pH is also detrimental for the growth of algae. In higher pH, acetic acid dissociates to form acetate ions causing inhibition to the growth of the cells (Wang and Wang, 1984). Microalgae are rich source of important metals. Accumulations of these elements are necessary for the proper function of their metabolism. Algal chlorophyll assimilate most of the elements such as iron, calcium and other minerals from the medium for the construction of their nucleic acids and other nucleic proteins (Pigott and Carr, 1971). Iron is a constituent of cytochromes and is a functional part of ferredoxin. It plays important role in synthesis of chlorophyll and nitrogen assimilation. Iron deficiency causes retardation in growth, reduction in photosynthetic activity, and chlorophyll content. Lack of iron is the reason for the bleaching and yellowing of algae (Becker, 1994). Magnesium (Mg) occupies the central position in structure of chlorophyll and therefore, cells loose chlorophyll in Mg deficient culture. Mg deficiency also disrupts cell division. It plays important role in aggression of ribosomes into functional unit (Becker, 1994). Role of Calcium is not known completely. However, it is believed to play important role in the maintenance of cytoplasmic membrane and the part of skeleton in certain algae (Becker, 1994). Zinc is essential for the normal functioning of enzyme systems within algae especially those, which catalyzes the algal photosynthesis and metabolism, for example acid phosphatase and alkaline phosphatase (Boyer and Brand, 1998). Low concentration of zinc may promote the multiplication of algae. However, higher concentration of Zn2+ may urge the nucleic acid to degrade and suppress both NADPH formation in the chloroplast and the cellular ATP level. Chlorophyll is the main impurity present in the carotene extracts. Chlorophyll may be 5–20 times the amount of carotene, depending on the nature of the extracting solvent. Presence of chlorophyll in carotene extracts gives an unattractive appearance causing decrease in stability of carotene to light. Chlorophyll can be removed by the principle that chlorophyll is normally soluble in varying degrees in most of the common non polar solvents such as petroleum ether, ether, chlorinated solvents, and alcohols except aqueous solvents. Contrary to this, esterified chlorophyll is insoluble in fat solvents but is soluble in polar solvents. The two ester group of chlorophyll could be saponified on reaction with KOH (present in miscible solvent such as methanol). Previously, chlorophyll was removed by saponification process on heating at 70 °C in 5% KOH in methanol for 5 min (Hu et al., 2013). On boiling this mixture, chlorophyll was rapidly saponified. Heat treatment reduces the surface tension and viscosity of the solvent. The solvent reaches to the active sites inside the matrix more easily at higher temperature. In addition, high temperature decreases the cell barrier by weakening integrity of the cell wall and membrane. As a result, heat treatment accelerates the mechanism of the diffusion of the solvent enhances its possibility to get in contact with bioactive compounds (Mohamad et al., 2010). The alkali treatment has no effect on carotene and xanthophylls. Thus, the present study aimed to investigate the effect of different physico-chemical parameters such as temperature, light, pH, carbon source on the cell growth characteristics of C. sorokiniana. Stoichiometric analysis was carried out to find the amount of CO2 capture and transformation into algal cellular constituents. Nutritional properties of algal biomass were speculated based on the proximate and metal analysis. Besides this, efforts were made to extract and quantify the astaxanthin, and b-carotene from the C. sorokiniana biomass.
359
2. Methods 2.1. Growth medium for C. sorokiniana The C. sorokiniana was grown in TAP, and TAP (-acetate) medium using glacial acetic acid and CO2 as carbon source, respectively (Kumar et al., 2013). Glacial acetic acid in the TAP medium was replaced by 1.5 mL HCl (37%) per liter (Kumar et al., 2014). The initial pH of the medium was found to be 7.3 ± 0.2. Optimization of physico-chemical parameters such as pH and nutrient were carried out in Erlenmeyer flasks. For autotrophic culture, air was sparged continuously with an aquarium pump at the flow rate of 0.33 vvm through one of the bottom side opening. Algal cultures were mixed continuously with the help of magnetic beads placed in the Erlenmeyer flask and multispin stirrer. Light source was compact fluorescent lamp (CFL) of 20 W, which was kept at the middle of the multispin stirrer. Temperature optimization was carried out in 250 mL double jacketed reactor. Temperature was maintained by circulating temperature controlled water through the outer jacket of the reactor. Algal cultures were placed in the inner jacket and mixed uniformly with magnetic beads and stirrer. Average light intensity falling on the surface of the double jacket reactor was adjusted to 100 lmol photons m2 s1. Light optimization was conducted in tissue culture flasks with flat sides to ensure uniform light distribution inside the culture. Photosynthetic active radiation (PAR) was adjusted using CFL by keeping them at different positions. 2.2. Dry cell weight estimation Cell concentration was determined as reported in APHA (1998). A sample of homogeneous algal cell suspension (1 mL) was collected from the photobioreactor. The biomass (pellet) was separated by centrifugation (5000g, 10 min, 4 °C) and washed three times with 0.85% w/v saline. This biomass was dried in a hot air oven at 80 ± 5 °C in pre weighed Aluminum (Al)-foil until a constant weight was obtained. Optical density (OD) was determined in spectrophotometer (Chemito). Biomass concentration was calculated using Eq. (1) by a calibration curve.
Biomass concentration ðmg mL1 Þ ¼ 0:28 OD682 nm
ð1Þ
2.3. CHNS determination Simultaneous determination of major components such as carbon, hydrogen, sulfur and nitrogen was carried out by combustion in CHNS analyzer (Elementar, vario Macro cube). Gas was detected and quantified using thermal conductivity detector (TCD). Helium (He) was used as a carrier gas at a flow rate of 600 mL min1. Relative percent of oxygen was calculated by deducting the relative percent of C, H, N, S from 100. 2.4. Determination of metal and proximate composition Proximate composition of biomass was determined using standard AOAC methods (AOAC, 1997). Total protein content was calculated by multiplying total nitrogen content by 6.25 (N 6.25), assuming algal protein contains 16% of nitrogen. Atomic absorption spectrophotometer (Perkin Elmer, AAnalyst 700) was used to determine the elements present in the algal biomass. One gram of lyophilized sample was placed in silica dish and incinerated over a burner. Incineration was stopped when smoke ceases. The dish was kept in muffle furnace at 550–600 °C. The dish was removed when light gray ash was obtained and cooled down to room temperature. HCl (10 mL) was added over the dish and heated over water bath for 30 min. Further 4 mL of HCl and Millipore water
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was added to dissolve the soluble salts and filtered using Whatman No. 41 filter paper. Filtrate was diluted to 50 mL and analyzed in atomic absorption spectroscopy (AAS) using Intensitron (Hollow Cathode) lamp. The absorbance of AAS was in flame mode. Quantification of elements was displayed in triplicate using standard known concentration dissolved in millipore water.
1
2.5. Extraction of pigments (astaxanthin, b-carotene)
l
Lyphilized algal biomass of 100 mg was dissolved in the 10 mL of extracting solvents acetone:hexane (1:4) followed by heating at 70 °C. One by third volume (3.3 mL) of 5% KOH dissolved in methanol was further added and solution was heated at 70 °C for 30 min. It resulted into formation of two layers, the bottom layer of polar solvents and upper layer of non polar solvents. Upper layer containing carotenoids was separated and analyzed in HPLC. 2.6. HPLC analysis Pigments present in the extracts were carried out in HPLC. An Agilent 1200 HPLC series (Agilent, Palo Alto, CA) equipped with a multi wavelength detector was used together with Agilent Eclipse-XDB C18 analytical column (5 lm, 250 4.6 mm i.d.) as stationary phase to perform the experiments. The chromatographic peaks were recorded at 450 nm. Two mobile solvents were employed (A, Acetonitrile; B, Isopropanol) following the gradient elution (1 min, 85% A; 19 min, 50% A; 21 min, 85% A) at the flow rate of 1.4 mL/min. Left and right column temperature were 30 °C. Samples were injected using a manual injection port (20 lL injection volume). A b-carotene and astaxanthin calibration curves (R2 = 0.97) were prepared using their standard (Sigma). Astaxanthin, and b-carotene were identified at retention time (tr) of 3.169 min, and 19.517 min, respectively. 2.7. Growth activation energy Arrhenius equation was used to determine the growth activation energy and Arrhenius constant.
lnet ¼ A expðEa =RTÞ
ð2Þ
After taking natural logarithm, Eq. (2) can be rewritten as Eq. (3)
ln lnet ¼ ln A Ea =RT
ð3Þ
where A is Arrhenius constant, Ea (kJ mol1) is growth activation energy, R is universal gas constant, which is equal to 8.314 J °K1 mol1, T (°K) is absolute temperature. 2.8. Cell growth kinetics constants Net specific growth rate was calculated from Eq. (4) (Kumar et al., 2014):
lnet ¼
ln N2 ln N1 ðt2 t 1 Þ
ð4Þ
where N2 and N1 were the biomass concentration at days t2 and t1 respectively. Net specific growth rate was taken in exponential phase. Doubling time was determined as follows (Eq. (5)):
td ¼
ln 2
l
ð5Þ
where, td is the doubling time. Minimum doubling time td(min) was calculated by replacing l by lmax. Generation time was calculated by taking reciprocal of td. Generation time is defined as the time interval for cells to divide. The Monod equation may be described as follows (Eq. (6)):
l¼
lmax S Ks þ S
where l, lmax and Ks are specific growth rate, maximum specific growth rate and substrate affinity constant, respectively. Ks is defined as the limiting substrate concentration (S) where l = 0.5 lmax. The values of kinetic parameters of Monod equation were determined by regression analysis of the linearized Lineweaver– Burk equation (Eq. (7)).
ð6Þ
¼
1
lmax
þ
Ks 1
lmax S
ð7Þ
3. Results and discussion 3.1. Effect of temperature Studies were conducted to find the suitable temperature for the growth of C. sorokiniana. Temperature was studied in the range of 20–45 °C. Most suitable temperature for the growth of this alga was found to be 30 °C. However, in the range of 30–40 °C, it had nearly similar growth profile (Fig. 1A). No growth of this alga was observed at 45 °C. The correlation of net specific growth rate and temperature was similar to other reported in the literature for Chlorella sp. KR-1(Sung et al., 1999). Previously, temperature range of 28–32 °C was found suitable for the growth of C. sorokiniana (Cordero et al., 2011). Temperature below the optimum was found to decrease the growth rate of the microorganism. At 20 °C, lag phase of one day was observed. However, the slope of log phase was steep. It showed the robustness of this strain for growing at colder climatic conditions. Plot of natural logarithm of specific growth and reciprocal of temperature had a bell shaped curve (Fig. 1B). Investigations were carried out to find out the suitability of Arrhenius equation to find out the effect of temperature on the biomass production. The values of Arrhenius constant (A), and growth activation energy (Ea) were determined using this model. Growth activation energy was calculated in the temperature range, when temperature had positive correlation with the growth. Eq. (3) shows a linear relationship between the natural logarithm of net specific growth and reciprocal of temperature. Constants were determined by curve fitting the experimental data. The Arrhenius equation was found to reasonably fit the experimental data as shown in inset of Fig. 1B (R2 = 0.97). Ea was found to be 7.08 kJ mol1 After incorporating the values of the . constants, Arrhenius equation (Eq. (3)) can be rewritten as Eq. (8).
ln lnet ¼ 3:21 7:08=RT
ð8Þ
Specific growth rate had positive correlation with temperature range from 20 to 30 °C. Scenedesmus sp. LX1 had Ea of 49.3 kJ mol1 (Xin et al., 2011). Chlorella vulgaris had activation energy of 24.5 kJ mol1 (Mayo, 1997). Low activation energy of this alga may signify its fast growing nature compared to other algae. 3.2. Effect of light intensity Light intensity plays important role for the growth of photosynthetic microorganism (Kumar et al., 2013). Effect of light intensity was investigated in flat tissue culture flasks. Flat tissue culture flasks were used in this study to ensure uniform availability of light for the algal cells. Growth characteristics were observed in the range of 0–250 lmol m2 s1 (Fig. 2A and B). Light intensity of 100 lmol m2 s1 was found most suitable for the growth of this microorganism (Fig. 2B). At lower light intensity (50 lmol m2 s1), linear increase in growth was obtained, which indicates that growth of cells were light limited. Contrary to this, at other higher light intensities, growth of cells reached to a saturation value. No light inhibition was observed up to the light intensity of 250 lmol m2 s1.
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K. Kumar et al. / Bioresource Technology 167 (2014) 358–366 Temperature (oC) 0.42
A
45
40
35
30
25
20
15
3.40
3.45
B
0.40 0.38
ln µnet
0.36 0.34
0.44 0.42 0.40
0.32 ln µnet
0.38
0.30
0.36 0.34 0.32 0.30 0.28
0.28
0.26 3.28
3.30
3.32
3.34
3.36
3.38
3.40
3.42
1/T × 10 -3 ( oK)
0.26 3.15
3.20
3.25
3.30
3.35
-3 o
1/T × 10 ( K)
Fig. 1. (A) Cell growth profile and (B) plot of net specific growth rate vs. temperatures using Chlorella sorokiniana. Error bar represents the SD for n = 3.
-2 -1
Light intensity (μmol m s ) 0
2.5
50
100
150
200
-2 -1 -2 -1
100 μmol m s
3.0
-2 -1
150 μmol m s
-1
2.0
-2 -1
200 μmol m s -2 -1 250 μmol m s Max. dwt
1.5
B
2.5
Net specific growth rate (d )
-1
Biomass concentration (g L )
250
A
50 μmol m s
1.0
0.5
2.0
1.5
1.0
0.5
0.0
0.0 0
10
20
30
40
50
60
70
Time (h)
0
50
100
150
200
250
300
-2 -1
Light intensity (μmol m s )
Fig. 2. (A) Cell growth profile and (B) plot of net specific growth rate and light intensity using Chlorella sorokiniana. Error bar represents the SD for n = 3.
3.3. Effect of initial medium pH Effect of pH on the cell growth profile of C. sorokiniana was conducted in two conditions: Mixotrophic condition (the medium was supplemented with acetic acid as carbon source), and Autotrophic condition (the CO2 was the only carbon source in the medium). In both the conditions, pH 7.5 was found favorable for the growth of this strain (Fig. 3A). At pH 7.5, maximum dry cell weight increased up to 1.4 g L1 in two days. Both the acidic and alkaline pH inhibits the growth of algae. Goldman et al. (1982) observed that C. vulgaris can grew up to pH 10.6, but the growth was adversely affected in alkaline pH. Similarly, Kumar et al. (2014) observed that acidic pH had inhibitory effect on the growth of C. sorokiniana. Increase in pH was observed with the advancement of growth period. In the pH range of 6 and 7.5, initially pH was found to increase to maximum, followed by its decline (Fig. 3B). Initial consumption of CO2 and glacial acetic acid may be the possible reasons for the increase in the pH. Decrease in pH after the exponential phase may be due to release of the CO2 (in cell’s respiration) and other acidic compounds in the medium. At initial pH 8, and pH 8.5, pH was increased continuously. Further, initial pH of 5.5 was found detrimental for the growth of this alga below which, no growth was observed.
In autotrophic growth condition, it was found that below pH 6.5, growth of the cell declines significantly (Fig. 3C). At initial pH 7.5, cell concentration of 1.18 g L1 was obtained in four days of cultivation. Contrary to mixotrophic condition, decrease in pH was observed, when CO2 was considered as the only carbon source in the medium. This may be due to acidification of medium because of air sparging. Sharp fall in pH was observed below initial pH of 6.5. However, slow and gradual decrease in the pH was found above initial pH 6.5 (Fig. 3D).
3.4. Effect of acetic acid The growth of C. sorokiniana was found out at the different concentrations of acetic acid in the range of 8.5–52.2 mM with 8.5 mM increment. Drop in pH due to addition of acetic acid concentration more than 17.4 mM was maintained using KOH solution. 34.8 mM glacial acetic acid was found to be most suitable for the growth of this strain. At this concentration net specific growth rate was increased by 1.35 times as compared to standard TAP medium containing 17.4 mM acetic acid (Fig. 4A and B). No substrate inhibition was observed up to the acetic acid concentration of 52.2 mM.
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Fig. 3. (A) Cell growth profile and (B) pH profile at different initial pH in the medium supplemented with glacial acidic acid as carbon source. (C) Cell growth profile and (D) pH profile at different initial pH in the medium sparged with air. Error bar represents the SD for n = 3.
Fig. 4. Effect of initial concentration of glacial acetic acid on the (A) cell growth profile and (B) net specific growth rate of C. sorokiniana. Error bar represents the SD for n = 3.
3.5. Cell growth kinetics The cell growth kinetics of C. sorokiniana was determined at most suitable physico-chemical conditions. The biomass and resid-
ual acetic acid profiles during the growth of C. sorokiniana in a batch system are depicted in Fig. 5A. Growth profile had three distinct phases namely: lag, log and stationary phase. Preliminary investigations were carried out to find out the suitability of Monod
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equation for biomass growth kinetics. Maximum specific growth rate (lmax), and saturation constant (Ks) were estimated using this model by regression analysis of Lineweaver–Burk linearized equation (Fig. 5B). Lineweaver–Burk plot was found to fit the growth profile of algae (R2 = 0.91). The values of different kinetic constants are tabulated in Table 1. The lmax, and Ks were 0.1 ± 0.01 h1 and 76 ± 8 mg L1, respectively using acetic acid as limiting as
substrate. The highest specific growth rate reported for this microorganism was 0.11 h1 (Cordero et al., 2011). The minimum doubling time (td(min)) was calculated with the help of lmax. The kinetic constant, lmax and td(min) show quick growing nature of the alga. This also gives an idea of the range of the dilution rate, which can be operated in continuous production of algal biomass using acetic acid as substrate. The Monod kinetic constant, Ks
3.0
2000
Biomass concentration (g L
) -1
-1
)
Dry cell weight Acetic acid
1500
2.0
1.5
1000
1.0 500 0.5
0.0
Acetate concentration (mg L
2.5
0 0
20
40
60
80
100
120
140
160
Time (h)
100
B
1/µ (h)
80
60
40
20
0 0.00
0.02
0.04
0.06
0.08
0.10
0.12
1 /S ( m g L -1 ) -1
8 .5
C
8 .0
Ln (Dwt)
7 .5
7 .0
6 .5
6 .0
5 .5 0
20
40
60
80
100
120
140
160
Tim e ( h) Fig. 5. (A) Cell growth and substrate concentration profiles, (B) Lineweaver–Burk plot, and (C) plot of natural logarithm of dry cell weight vs. time of C. sorokiniana (Experimental conditions: average light intensity falling on the surface of BioEngineering AG photobioreactor was 300 lmol m2 s1. Temperature – 30 °C, Initial pH – 7.5; Medium composition std. TAP medium with 34.8 mM acetic acid; Error bar represents the SD for n = 3).
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Table 1 Kinetic constants of C. sorokiniana in BioEngineering AG photobioreactor equipped with external light jacket. Kinetic parameters
Kinetic constants
Maximum specific growth rate (lmax) Minimum doubling time (td(min)) Substrate affinity constant (Ks) Net specific growth rate (lnet) Doubling time (td) Generation time (G)
0.1 ± 0.01 h1 6.9 ± 0.6 h 76 ± 8 mg L1 0.01 ± 0.0003 h1 69.3 ± 20 h 0.014 ± 0.003 h1
The data are given as mean ± SD, n = 3.
represents the substrate concentration required to achieve 50% of the maximum growth rate, therefore it suggest guidelines for maintaining the most suitable acetic concentration in the feed. This becomes more important in case of wastewater treatment, where acetic acid concentration is found in the similar range. Another constant, net specific growth rate, lnet was calculated from the slope of natural logarithm of dry cell weight vs. time plot (Fig. 5C). The doubling time, td of 69.3 ± 20 h and generation time of 0.014 ± 0.003 h1, further confirms the fast growing nature of the alga and a potential microorganism for wastewater treatment. 3.6. CHNS analysis C, H, N and S content of C. sorokiniana algal biomass were determined at different experimental conditions as shown in Table 2. Results were found similar with previous reported data (Rizzo et al., 2013; Friis et al., 1998). Elemental composition of algal biomass of Chlorella sp. with respect to C, H, N and S was reported as 46.1–50.1%, 6.1–7.4%, 6.7–7.9%,
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Cell growth kinetics of Chlorella sorokiniana and nutritional values of its biomass Kanhaiya Kumar, Chitralekha Nag Dasgupta, Debabrata Das ⇑ Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India
h i g h l i g h t s Physico-chemical parameters were optimized for the growth of C. sorokiniana. Arrhenius growth activation energy was calculated to show its fast growing nature. Monod model was used to study the growth kinetics of this microorganism. Stoichiometric analysis was performed to measure CO2 intake and biomass synthesis. Astaxanthin and b-carotene were separated from chlorophyll contamination.
a r t i c l e
i n f o
Article history: Received 11 May 2014 Received in revised form 29 May 2014 Accepted 31 May 2014 Available online 16 June 2014 Keywords: Temperature pH Light Elemental analysis Pigment extraction
a b s t r a c t The present study investigates the effects of different physico-chemical parameters for the growth of Chlorella sorokiniana and subsequently determination of nutritional values of its biomass. Most suitable temperature, light intensity, pH, and acetic acid concentration were 30 °C, 100 lmol m2 s1, pH 7.5, and 34.8 mM, respectively for the growth of this microorganism. Arrhenius growth activation energy, Ea was calculated as 7.08 kJ mol1. Monod kinetics constants: maximum specific growth rate (lmax) and substrate (acetic acid) affinity coefficient (Ks) were determined as 0.1 ± 0.01 h1 and 76 ± 8 mg L1, respectively. Stoichiometric analysis revealed the capture of 1.83 g CO2 and release of 1.9 g O2 for 1 g algal biomass synthesis. Algal biomass of C. sorokiniana was found rich in protein and several important minerals such as Mg, Ca, and Fe. Astaxanthin and b-carotene were extracted and quantified using high performance liquid chromatography. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction World is facing several challenges such as inadequate availability of food, feed and fuels. Global warming due to greenhouse gases like, CO2 is a major concern (Kumar and Das, 2014). Exploring the algal technology is a promising area, which can support the supply of these demands. Microalgae can transform light energy into chemical energy to meet the future energy demand (e.g. biodiesel, biohydrogen) and they can produce a great variety of useful metabolites (Kumar et al., 2013; Xin et al., 2011). It is a promising source of several pigments such as lutein, b-carotene, and astaxanthin (Kumar et al., 2014; Cordero et al., 2011). These pigments are known for their excellent antioxidant and pharmaceutical properties. In addition, algal biomass has high nutritional values as it is
⇑ Corresponding author. Tel.: +91 03222 283758; fax: +91 3222 255303. E-mail address: [email protected] (D. Das). http://dx.doi.org/10.1016/j.biortech.2014.05.118 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
rich source of protein, carbohydrate, essential fatty acids, dietary fibers, minerals etc. Different physio-chemical parameters such as pH, temperature, light, different nutrients are governing the activities and growth rates of microalgae when they are grown in different carbon sources like CO2 and acetic acid. At low light intensity, photosynthesis becomes the limiting factor. The light intensity above the saturation level inhibits the growth by photoinhibition and excess excitation energy is dissipated as fluorescence or heat through non-photochemical quenching (NPQ) (Yamakawa and Itoh, 2013). The low temperature reduces many cellular activities such as the ability to use light energy by limiting the electron transport chain etc. (Falk et al., 1990). Photosystem II is also very thermolabile, causing inhibition of photosynthesis at higher temperature (Fork et al., 1979). Photorespiration was found to increase with the increase in temperature (Berry and Raison, 1981). In addition, temperature can also affect the cytosolic pH and other physical processes (Raven and Geider, 1978). Mostly the microalgae
K. Kumar et al. / Bioresource Technology 167 (2014) 358–366
maintain neutral or slightly alkaline cytosolic pH, as many enzymes are highly pH dependent and become inactive in acidic pH (Gimmler, 2001). Therefore, external pH plays important role for maintaining the cytosolic pH. In addition, it has been found that pH is the major determinant for the availability of the carbonaceous species in the broth for the growth of algae. High alkaline pH is also detrimental for the growth of algae. In higher pH, acetic acid dissociates to form acetate ions causing inhibition to the growth of the cells (Wang and Wang, 1984). Microalgae are rich source of important metals. Accumulations of these elements are necessary for the proper function of their metabolism. Algal chlorophyll assimilate most of the elements such as iron, calcium and other minerals from the medium for the construction of their nucleic acids and other nucleic proteins (Pigott and Carr, 1971). Iron is a constituent of cytochromes and is a functional part of ferredoxin. It plays important role in synthesis of chlorophyll and nitrogen assimilation. Iron deficiency causes retardation in growth, reduction in photosynthetic activity, and chlorophyll content. Lack of iron is the reason for the bleaching and yellowing of algae (Becker, 1994). Magnesium (Mg) occupies the central position in structure of chlorophyll and therefore, cells loose chlorophyll in Mg deficient culture. Mg deficiency also disrupts cell division. It plays important role in aggression of ribosomes into functional unit (Becker, 1994). Role of Calcium is not known completely. However, it is believed to play important role in the maintenance of cytoplasmic membrane and the part of skeleton in certain algae (Becker, 1994). Zinc is essential for the normal functioning of enzyme systems within algae especially those, which catalyzes the algal photosynthesis and metabolism, for example acid phosphatase and alkaline phosphatase (Boyer and Brand, 1998). Low concentration of zinc may promote the multiplication of algae. However, higher concentration of Zn2+ may urge the nucleic acid to degrade and suppress both NADPH formation in the chloroplast and the cellular ATP level. Chlorophyll is the main impurity present in the carotene extracts. Chlorophyll may be 5–20 times the amount of carotene, depending on the nature of the extracting solvent. Presence of chlorophyll in carotene extracts gives an unattractive appearance causing decrease in stability of carotene to light. Chlorophyll can be removed by the principle that chlorophyll is normally soluble in varying degrees in most of the common non polar solvents such as petroleum ether, ether, chlorinated solvents, and alcohols except aqueous solvents. Contrary to this, esterified chlorophyll is insoluble in fat solvents but is soluble in polar solvents. The two ester group of chlorophyll could be saponified on reaction with KOH (present in miscible solvent such as methanol). Previously, chlorophyll was removed by saponification process on heating at 70 °C in 5% KOH in methanol for 5 min (Hu et al., 2013). On boiling this mixture, chlorophyll was rapidly saponified. Heat treatment reduces the surface tension and viscosity of the solvent. The solvent reaches to the active sites inside the matrix more easily at higher temperature. In addition, high temperature decreases the cell barrier by weakening integrity of the cell wall and membrane. As a result, heat treatment accelerates the mechanism of the diffusion of the solvent enhances its possibility to get in contact with bioactive compounds (Mohamad et al., 2010). The alkali treatment has no effect on carotene and xanthophylls. Thus, the present study aimed to investigate the effect of different physico-chemical parameters such as temperature, light, pH, carbon source on the cell growth characteristics of C. sorokiniana. Stoichiometric analysis was carried out to find the amount of CO2 capture and transformation into algal cellular constituents. Nutritional properties of algal biomass were speculated based on the proximate and metal analysis. Besides this, efforts were made to extract and quantify the astaxanthin, and b-carotene from the C. sorokiniana biomass.
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2. Methods 2.1. Growth medium for C. sorokiniana The C. sorokiniana was grown in TAP, and TAP (-acetate) medium using glacial acetic acid and CO2 as carbon source, respectively (Kumar et al., 2013). Glacial acetic acid in the TAP medium was replaced by 1.5 mL HCl (37%) per liter (Kumar et al., 2014). The initial pH of the medium was found to be 7.3 ± 0.2. Optimization of physico-chemical parameters such as pH and nutrient were carried out in Erlenmeyer flasks. For autotrophic culture, air was sparged continuously with an aquarium pump at the flow rate of 0.33 vvm through one of the bottom side opening. Algal cultures were mixed continuously with the help of magnetic beads placed in the Erlenmeyer flask and multispin stirrer. Light source was compact fluorescent lamp (CFL) of 20 W, which was kept at the middle of the multispin stirrer. Temperature optimization was carried out in 250 mL double jacketed reactor. Temperature was maintained by circulating temperature controlled water through the outer jacket of the reactor. Algal cultures were placed in the inner jacket and mixed uniformly with magnetic beads and stirrer. Average light intensity falling on the surface of the double jacket reactor was adjusted to 100 lmol photons m2 s1. Light optimization was conducted in tissue culture flasks with flat sides to ensure uniform light distribution inside the culture. Photosynthetic active radiation (PAR) was adjusted using CFL by keeping them at different positions. 2.2. Dry cell weight estimation Cell concentration was determined as reported in APHA (1998). A sample of homogeneous algal cell suspension (1 mL) was collected from the photobioreactor. The biomass (pellet) was separated by centrifugation (5000g, 10 min, 4 °C) and washed three times with 0.85% w/v saline. This biomass was dried in a hot air oven at 80 ± 5 °C in pre weighed Aluminum (Al)-foil until a constant weight was obtained. Optical density (OD) was determined in spectrophotometer (Chemito). Biomass concentration was calculated using Eq. (1) by a calibration curve.
Biomass concentration ðmg mL1 Þ ¼ 0:28 OD682 nm
ð1Þ
2.3. CHNS determination Simultaneous determination of major components such as carbon, hydrogen, sulfur and nitrogen was carried out by combustion in CHNS analyzer (Elementar, vario Macro cube). Gas was detected and quantified using thermal conductivity detector (TCD). Helium (He) was used as a carrier gas at a flow rate of 600 mL min1. Relative percent of oxygen was calculated by deducting the relative percent of C, H, N, S from 100. 2.4. Determination of metal and proximate composition Proximate composition of biomass was determined using standard AOAC methods (AOAC, 1997). Total protein content was calculated by multiplying total nitrogen content by 6.25 (N 6.25), assuming algal protein contains 16% of nitrogen. Atomic absorption spectrophotometer (Perkin Elmer, AAnalyst 700) was used to determine the elements present in the algal biomass. One gram of lyophilized sample was placed in silica dish and incinerated over a burner. Incineration was stopped when smoke ceases. The dish was kept in muffle furnace at 550–600 °C. The dish was removed when light gray ash was obtained and cooled down to room temperature. HCl (10 mL) was added over the dish and heated over water bath for 30 min. Further 4 mL of HCl and Millipore water
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was added to dissolve the soluble salts and filtered using Whatman No. 41 filter paper. Filtrate was diluted to 50 mL and analyzed in atomic absorption spectroscopy (AAS) using Intensitron (Hollow Cathode) lamp. The absorbance of AAS was in flame mode. Quantification of elements was displayed in triplicate using standard known concentration dissolved in millipore water.
1
2.5. Extraction of pigments (astaxanthin, b-carotene)
l
Lyphilized algal biomass of 100 mg was dissolved in the 10 mL of extracting solvents acetone:hexane (1:4) followed by heating at 70 °C. One by third volume (3.3 mL) of 5% KOH dissolved in methanol was further added and solution was heated at 70 °C for 30 min. It resulted into formation of two layers, the bottom layer of polar solvents and upper layer of non polar solvents. Upper layer containing carotenoids was separated and analyzed in HPLC. 2.6. HPLC analysis Pigments present in the extracts were carried out in HPLC. An Agilent 1200 HPLC series (Agilent, Palo Alto, CA) equipped with a multi wavelength detector was used together with Agilent Eclipse-XDB C18 analytical column (5 lm, 250 4.6 mm i.d.) as stationary phase to perform the experiments. The chromatographic peaks were recorded at 450 nm. Two mobile solvents were employed (A, Acetonitrile; B, Isopropanol) following the gradient elution (1 min, 85% A; 19 min, 50% A; 21 min, 85% A) at the flow rate of 1.4 mL/min. Left and right column temperature were 30 °C. Samples were injected using a manual injection port (20 lL injection volume). A b-carotene and astaxanthin calibration curves (R2 = 0.97) were prepared using their standard (Sigma). Astaxanthin, and b-carotene were identified at retention time (tr) of 3.169 min, and 19.517 min, respectively. 2.7. Growth activation energy Arrhenius equation was used to determine the growth activation energy and Arrhenius constant.
lnet ¼ A expðEa =RTÞ
ð2Þ
After taking natural logarithm, Eq. (2) can be rewritten as Eq. (3)
ln lnet ¼ ln A Ea =RT
ð3Þ
where A is Arrhenius constant, Ea (kJ mol1) is growth activation energy, R is universal gas constant, which is equal to 8.314 J °K1 mol1, T (°K) is absolute temperature. 2.8. Cell growth kinetics constants Net specific growth rate was calculated from Eq. (4) (Kumar et al., 2014):
lnet ¼
ln N2 ln N1 ðt2 t 1 Þ
ð4Þ
where N2 and N1 were the biomass concentration at days t2 and t1 respectively. Net specific growth rate was taken in exponential phase. Doubling time was determined as follows (Eq. (5)):
td ¼
ln 2
l
ð5Þ
where, td is the doubling time. Minimum doubling time td(min) was calculated by replacing l by lmax. Generation time was calculated by taking reciprocal of td. Generation time is defined as the time interval for cells to divide. The Monod equation may be described as follows (Eq. (6)):
l¼
lmax S Ks þ S
where l, lmax and Ks are specific growth rate, maximum specific growth rate and substrate affinity constant, respectively. Ks is defined as the limiting substrate concentration (S) where l = 0.5 lmax. The values of kinetic parameters of Monod equation were determined by regression analysis of the linearized Lineweaver– Burk equation (Eq. (7)).
ð6Þ
¼
1
lmax
þ
Ks 1
lmax S
ð7Þ
3. Results and discussion 3.1. Effect of temperature Studies were conducted to find the suitable temperature for the growth of C. sorokiniana. Temperature was studied in the range of 20–45 °C. Most suitable temperature for the growth of this alga was found to be 30 °C. However, in the range of 30–40 °C, it had nearly similar growth profile (Fig. 1A). No growth of this alga was observed at 45 °C. The correlation of net specific growth rate and temperature was similar to other reported in the literature for Chlorella sp. KR-1(Sung et al., 1999). Previously, temperature range of 28–32 °C was found suitable for the growth of C. sorokiniana (Cordero et al., 2011). Temperature below the optimum was found to decrease the growth rate of the microorganism. At 20 °C, lag phase of one day was observed. However, the slope of log phase was steep. It showed the robustness of this strain for growing at colder climatic conditions. Plot of natural logarithm of specific growth and reciprocal of temperature had a bell shaped curve (Fig. 1B). Investigations were carried out to find out the suitability of Arrhenius equation to find out the effect of temperature on the biomass production. The values of Arrhenius constant (A), and growth activation energy (Ea) were determined using this model. Growth activation energy was calculated in the temperature range, when temperature had positive correlation with the growth. Eq. (3) shows a linear relationship between the natural logarithm of net specific growth and reciprocal of temperature. Constants were determined by curve fitting the experimental data. The Arrhenius equation was found to reasonably fit the experimental data as shown in inset of Fig. 1B (R2 = 0.97). Ea was found to be 7.08 kJ mol1 After incorporating the values of the . constants, Arrhenius equation (Eq. (3)) can be rewritten as Eq. (8).
ln lnet ¼ 3:21 7:08=RT
ð8Þ
Specific growth rate had positive correlation with temperature range from 20 to 30 °C. Scenedesmus sp. LX1 had Ea of 49.3 kJ mol1 (Xin et al., 2011). Chlorella vulgaris had activation energy of 24.5 kJ mol1 (Mayo, 1997). Low activation energy of this alga may signify its fast growing nature compared to other algae. 3.2. Effect of light intensity Light intensity plays important role for the growth of photosynthetic microorganism (Kumar et al., 2013). Effect of light intensity was investigated in flat tissue culture flasks. Flat tissue culture flasks were used in this study to ensure uniform availability of light for the algal cells. Growth characteristics were observed in the range of 0–250 lmol m2 s1 (Fig. 2A and B). Light intensity of 100 lmol m2 s1 was found most suitable for the growth of this microorganism (Fig. 2B). At lower light intensity (50 lmol m2 s1), linear increase in growth was obtained, which indicates that growth of cells were light limited. Contrary to this, at other higher light intensities, growth of cells reached to a saturation value. No light inhibition was observed up to the light intensity of 250 lmol m2 s1.
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A
45
40
35
30
25
20
15
3.40
3.45
B
0.40 0.38
ln µnet
0.36 0.34
0.44 0.42 0.40
0.32 ln µnet
0.38
0.30
0.36 0.34 0.32 0.30 0.28
0.28
0.26 3.28
3.30
3.32
3.34
3.36
3.38
3.40
3.42
1/T × 10 -3 ( oK)
0.26 3.15
3.20
3.25
3.30
3.35
-3 o
1/T × 10 ( K)
Fig. 1. (A) Cell growth profile and (B) plot of net specific growth rate vs. temperatures using Chlorella sorokiniana. Error bar represents the SD for n = 3.
-2 -1
Light intensity (μmol m s ) 0
2.5
50
100
150
200
-2 -1 -2 -1
100 μmol m s
3.0
-2 -1
150 μmol m s
-1
2.0
-2 -1
200 μmol m s -2 -1 250 μmol m s Max. dwt
1.5
B
2.5
Net specific growth rate (d )
-1
Biomass concentration (g L )
250
A
50 μmol m s
1.0
0.5
2.0
1.5
1.0
0.5
0.0
0.0 0
10
20
30
40
50
60
70
Time (h)
0
50
100
150
200
250
300
-2 -1
Light intensity (μmol m s )
Fig. 2. (A) Cell growth profile and (B) plot of net specific growth rate and light intensity using Chlorella sorokiniana. Error bar represents the SD for n = 3.
3.3. Effect of initial medium pH Effect of pH on the cell growth profile of C. sorokiniana was conducted in two conditions: Mixotrophic condition (the medium was supplemented with acetic acid as carbon source), and Autotrophic condition (the CO2 was the only carbon source in the medium). In both the conditions, pH 7.5 was found favorable for the growth of this strain (Fig. 3A). At pH 7.5, maximum dry cell weight increased up to 1.4 g L1 in two days. Both the acidic and alkaline pH inhibits the growth of algae. Goldman et al. (1982) observed that C. vulgaris can grew up to pH 10.6, but the growth was adversely affected in alkaline pH. Similarly, Kumar et al. (2014) observed that acidic pH had inhibitory effect on the growth of C. sorokiniana. Increase in pH was observed with the advancement of growth period. In the pH range of 6 and 7.5, initially pH was found to increase to maximum, followed by its decline (Fig. 3B). Initial consumption of CO2 and glacial acetic acid may be the possible reasons for the increase in the pH. Decrease in pH after the exponential phase may be due to release of the CO2 (in cell’s respiration) and other acidic compounds in the medium. At initial pH 8, and pH 8.5, pH was increased continuously. Further, initial pH of 5.5 was found detrimental for the growth of this alga below which, no growth was observed.
In autotrophic growth condition, it was found that below pH 6.5, growth of the cell declines significantly (Fig. 3C). At initial pH 7.5, cell concentration of 1.18 g L1 was obtained in four days of cultivation. Contrary to mixotrophic condition, decrease in pH was observed, when CO2 was considered as the only carbon source in the medium. This may be due to acidification of medium because of air sparging. Sharp fall in pH was observed below initial pH of 6.5. However, slow and gradual decrease in the pH was found above initial pH 6.5 (Fig. 3D).
3.4. Effect of acetic acid The growth of C. sorokiniana was found out at the different concentrations of acetic acid in the range of 8.5–52.2 mM with 8.5 mM increment. Drop in pH due to addition of acetic acid concentration more than 17.4 mM was maintained using KOH solution. 34.8 mM glacial acetic acid was found to be most suitable for the growth of this strain. At this concentration net specific growth rate was increased by 1.35 times as compared to standard TAP medium containing 17.4 mM acetic acid (Fig. 4A and B). No substrate inhibition was observed up to the acetic acid concentration of 52.2 mM.
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Fig. 3. (A) Cell growth profile and (B) pH profile at different initial pH in the medium supplemented with glacial acidic acid as carbon source. (C) Cell growth profile and (D) pH profile at different initial pH in the medium sparged with air. Error bar represents the SD for n = 3.
Fig. 4. Effect of initial concentration of glacial acetic acid on the (A) cell growth profile and (B) net specific growth rate of C. sorokiniana. Error bar represents the SD for n = 3.
3.5. Cell growth kinetics The cell growth kinetics of C. sorokiniana was determined at most suitable physico-chemical conditions. The biomass and resid-
ual acetic acid profiles during the growth of C. sorokiniana in a batch system are depicted in Fig. 5A. Growth profile had three distinct phases namely: lag, log and stationary phase. Preliminary investigations were carried out to find out the suitability of Monod
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equation for biomass growth kinetics. Maximum specific growth rate (lmax), and saturation constant (Ks) were estimated using this model by regression analysis of Lineweaver–Burk linearized equation (Fig. 5B). Lineweaver–Burk plot was found to fit the growth profile of algae (R2 = 0.91). The values of different kinetic constants are tabulated in Table 1. The lmax, and Ks were 0.1 ± 0.01 h1 and 76 ± 8 mg L1, respectively using acetic acid as limiting as
substrate. The highest specific growth rate reported for this microorganism was 0.11 h1 (Cordero et al., 2011). The minimum doubling time (td(min)) was calculated with the help of lmax. The kinetic constant, lmax and td(min) show quick growing nature of the alga. This also gives an idea of the range of the dilution rate, which can be operated in continuous production of algal biomass using acetic acid as substrate. The Monod kinetic constant, Ks
3.0
2000
Biomass concentration (g L
) -1
-1
)
Dry cell weight Acetic acid
1500
2.0
1.5
1000
1.0 500 0.5
0.0
Acetate concentration (mg L
2.5
0 0
20
40
60
80
100
120
140
160
Time (h)
100
B
1/µ (h)
80
60
40
20
0 0.00
0.02
0.04
0.06
0.08
0.10
0.12
1 /S ( m g L -1 ) -1
8 .5
C
8 .0
Ln (Dwt)
7 .5
7 .0
6 .5
6 .0
5 .5 0
20
40
60
80
100
120
140
160
Tim e ( h) Fig. 5. (A) Cell growth and substrate concentration profiles, (B) Lineweaver–Burk plot, and (C) plot of natural logarithm of dry cell weight vs. time of C. sorokiniana (Experimental conditions: average light intensity falling on the surface of BioEngineering AG photobioreactor was 300 lmol m2 s1. Temperature – 30 °C, Initial pH – 7.5; Medium composition std. TAP medium with 34.8 mM acetic acid; Error bar represents the SD for n = 3).
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Table 1 Kinetic constants of C. sorokiniana in BioEngineering AG photobioreactor equipped with external light jacket. Kinetic parameters
Kinetic constants
Maximum specific growth rate (lmax) Minimum doubling time (td(min)) Substrate affinity constant (Ks) Net specific growth rate (lnet) Doubling time (td) Generation time (G)
0.1 ± 0.01 h1 6.9 ± 0.6 h 76 ± 8 mg L1 0.01 ± 0.0003 h1 69.3 ± 20 h 0.014 ± 0.003 h1
The data are given as mean ± SD, n = 3.
represents the substrate concentration required to achieve 50% of the maximum growth rate, therefore it suggest guidelines for maintaining the most suitable acetic concentration in the feed. This becomes more important in case of wastewater treatment, where acetic acid concentration is found in the similar range. Another constant, net specific growth rate, lnet was calculated from the slope of natural logarithm of dry cell weight vs. time plot (Fig. 5C). The doubling time, td of 69.3 ± 20 h and generation time of 0.014 ± 0.003 h1, further confirms the fast growing nature of the alga and a potential microorganism for wastewater treatment. 3.6. CHNS analysis C, H, N and S content of C. sorokiniana algal biomass were determined at different experimental conditions as shown in Table 2. Results were found similar with previous reported data (Rizzo et al., 2013; Friis et al., 1998). Elemental composition of algal biomass of Chlorella sp. with respect to C, H, N and S was reported as 46.1–50.1%, 6.1–7.4%, 6.7–7.9%,
